Sanaria's platform technology is production of aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ). This technology has produced three products administered by needle and syringe, all of which are in clinical trials - PfSPZ Vaccine (radiation attenuated PfSPZ), PfSPZ Challenge (infectious PfSPZ for controlled human malaria infection [CHMI]) and PfSPZ-CVac (PfSPZ Challenge given with antimalarial drugs as a vaccine). PfSPZ Vaccine protected 100% (6/6) volunteers against CHMI in a clinical trial at the NIH. The reproducibility of the NIH findings, durability of protection, protection against heterologous Pf parasites, and reduced numbers of doses with altered vaccine regimens are being assessed in 6 clinical trials at 7 sites in the U.S., Europe, and Africa. By March 2014, our Malian collaborators had injected >100 volunteers twice at a 4 week interval by direct venous inoculation (DVI); the injections have gone perfectly and been extremely well tolerated. 7 clinical trials of PfSPZ Challenge have been conducted in 7 countries; 100% infection was achieved in five of the trials. A trial of the PfSPZ-CVac approach begins in Germany in May 2014. There is tremendous international support for our whole PfSPZ approach for malaria vaccines and products for CHMI. Sanaria's products rely on production of PfSPZ in aseptic mosquitoes. Our Phase I SBIR project proposed developing a method for producing PfSPZ in culture (in vitro) that would eliminate the need for mosquitoes. We estimated this would reduce cost of producing vaccine by ~80%. The Phase I SBIR was successful. We developed methods for producing Pf oocysts in vitro with an efficiency 39 times greater than when we produce oocysts in mosquitoes. We repeatedly produced PfSPZ in vitro that invaded and developed to mature 6 day liver stage schizonts expressing Pf merozoite surface protein 1 with the same efficiency as PfSPZ freshly dissected from mosquitoes. This is a first in the history of malaria research. Although we will significantly improve efficiency and quality of in vitro production of PfSPZ in Phase II, we could now actually manufacture enough PfSPZ in vitro to support a CHMI clinical trial of PfSPZ Challenge-in vitro. In this Phase II projet we will optimize methods for manufacture of clinical grade PfSPZ in vitro. We will use 3D cell culture technologies to fully optimize methods for production of PfSPZ in vitro, and then demonstrate these PfSPZ can reproducibly invade and develop in human hepatocytes in vitro and in vivo, and complete the Pf life cycle. This will be done in vivo using human liver chimeric mice transfused with human blood. We will establish a method for purifying PfSPZ and an assay for quantifying purity. Our manufacturing, quality, and regulatory teams will ensure all reagents and processes are compliant with cGMPs and adequate for manufacturing, conduct engineering runs, and submit a pre-IND package to FDA for a CHMI clinical trial of PfSPZ Challenge-in vitro to demonstrate infectivity to humans, thereby establishing a rationale for moving from mosquito- to in vitro-produced PfSPZ for our vaccines.